Antigenic localities in the tissues of Metagonimus yokogawai observed by immunogoldlabeling method]

In order to determine the antigenic localization in the tissues of the adult Metagonimus yokogawai, immunogoldlabeling method was applied using serum immunoglobulins(IgG) of cats which were infected with isolated metacercariae from Plecoglossus altivelis. The sectioned worm tissue was embedded in Lowicryl HM 20 medium and stained with infected serum IgG and protein A gold complex(particle size: 12 nm). It was observed by electron microscopy at each tissue of the worm. The gold particles were observed on the tegumental syncytium as well as cytoplasm of tegumental cells and epithelial lamella of the caecum. The gold particles were not observed on the basal lamina of the tegument, interstitial matrix of the parenchyma, the muscle tissue and mitochondria of the tegument. The gold particles were specifically labeled in the secretory granules in the vitelline cells. They were also labeled on the lumen of bladder and egg shell. The above findings showed that antigenic materials in the tissue of adult worms were specifically concentrated on the tegumental syncytium as well as cytoplasm of tegumental cells and epithelial lamella of the caecum.     

Antigenic localities in the tissues of Paragonimus westermani by developmental stages using immunogold labeling method]

In order to observe the antigenic localization in the tissues of Paragonimus westermani of developmental stages, immunogold labeling method was applied using serum of the cats which were infected with isolated metacercariae from Cambaroides similis. The sectioned worm tissues from each developmental stage were embedded in Lowicryl HM 20 medium, stained with infected serum IgG and protein A gold complex (particle size: 12 nm) and observed by electron microscopy. In the young adult worm tissue of 4 weeks after infection with metacercariae, the gold particles were specifically concentrated on the tegumental syncytium and cytoplasm of the tegumental cells as well as the secretory granules in the parenchymal tissue. The antigenic materials in the adult worm tissue were specifically concentrated on the secretory granules in the parenchymal tissue, the cytoplasm between granules in the vitelline gland and the epithelial lamella in the lumen of the caecum.     

Antigenic localities in the tissues of Metagonimus yokogawai in the period of growth]

In order to observe the antigenic localization in the tissues of Metagonimus yokogawai in growth stages, immunogoldlabeling method was applied to using serum of the cat which infected with isolated metacercariae from Plecoglossus altivelis. The sectioned worm tissues from each growth stages were embedded in Lowicryl HM 20 medium, stained with infected serum IgG and protein A gold complex (particle size: 12 nm) and observed by electron microscopy. In the worm tissues of all experimental groups, the gold particles were specifically concentrated on the tegumental syncytium and cytoplasm of the tegumental cell as well as the secretory granules in the parenchymal tissue. In the 16th and 20th week grown worm tissues, the gold particles were specifically concentrated on the vesicles in the tegumental syncytium and cytoplasm of the tegumental cell. The gold particles were specifically concentrated on the caecal epithelia of the 4th, 8th and 12th week growth groups but slightly concentrated on those of the 16th and 20th week.     

A study on the body fluid antigen of Clonorchis sinensis using immunogold labeling method

In order to observe the antigenic localization in the tissues of the adult Clonorchis sinensis, immunogold labeling method was applied using serum immunoglobulins (IgG) of either worm-infected rabbits (group I) or antigen-immunized rabbits (group II) (by the body fluid obtained from the adult worms). The electron micrographs of the sectioned worm tissue antigens, embedded in Lowicryl HM 20 medium and stained with protein A-gold complex (particle size: 12 nm), were compared between the group I and group II. The gold particles were observed in the interstitial matrix of the worm parenchyma, the epithelial lamellae of the cecum, and the cecal lumen both in group I and II. But the particles were in general more concentrated in group II. The gold particles were not observed on the basal lamina of the tegument or on vitelline glands in group I, while they were highly concentrated on those areas in group II. There were also differences in the antigenicity of interstitial matrix(reacted with group I IgG) and head part(reacted with group II IgG) of the sperm cells in the seminal receptacle. Conclusively, it is suggested that the substances comprising the basal lamina of the tegument or vitelline glands act as specific antigens reacting with antigen(body fluid) immunized rabbit IgG. On the other hand, the substances in the cecal lumen and cecal epithelial lamellae are thought to be the specific antigen that react with the worm-infected rabbit IgG.     

Fasciola hepatica: development of monoclonal antibodies against somatic antigens and their characterization by ultrastructural localization of antibody binding

A series of monoclonal antibodies was prepared against tegumental and internal antigens of Fasciola hepatica by immunizing mice with whole adult-fluke homogenates prior to harvesting the splenic lymphocytes for fusion. Preliminary screening by the Indirect Fluorescent Antibody technique indicated the occurrence of discrete groups of monoclonals differing from one another in tissue-specificity but within which IFA labelling patterns were fairly consistent. Representative hybridomas for 5 of these groups were stabilized and used to produce ascites fluid in mice. By application of an immunogold labelling technique it was possible to map the distribution of antigens for which each monoclonal antibody had affinity throughout the tissues of 4-week and 12-week flukes. Several monoclonals specifically labelled antigenic determinants on the important tegumental antigen T1. However the distribution of gold colloid labelling suggested that epitopes other than that normally exposed to the infected host were recognized; and several monoclonals specifically attached to T1 antigen in the tegument of juvenile worms only. The glycocalyx of the gut and excretory system of flukes shared T1 antigenicity with the tegument. Monoclonal antibodies were produced against an internal immunogen associated with ribosomes and heterochromatin in active protein-producing cells, and against interstitial material of adult flukes. Monoclonals against antigens in parenchymal cell cytoplasm and in mature vitelline cells were recognized but the corresponding hybridomas were not stabilized.     

Scanning electron microscopy of in vitro serum-mediated destruction of juvenile Fasciola hepatica

The tegumental surface of immature Fasciola hepatica was damaged when incubated in vitro with serum collected from an experimentally infected calf. Degeneration of the tegumental surface was observed by scanning electron microscopy (SEM) at 4 hr. after incubation. Decomposition was observed 8 to 12 hr after incubation and complete destruction of the tegument occurred by 16 hr. The flukes became inactive after 8 to 12 hr of incubation. None of the above findings were observed for the tegument of flukes incubated in tissue culture media or in media containing normal calf serum and the trematodes remained motile throughout the incubation period. Latex particles were used as an immunological marker for SEM studies to determine if gamma globulin could be responsible for the observed changes and, if so, the site of antibody attachment. The coated latex particles covered the entire surface of flukes recovered from mice 5 days after infection with metacercariae. In contrast, latex particles coated with either normal gamma globulin or gamma globulin from serum of the experimentally infected calf that had been adsorbed with disrupted adult flukes were not attached to the surface of the flukes. Absorption of the serum with disrupted, adult flukes decreased the concentrations of immunoglobulins (Ig)G1 and G2 whereas IgA and IgM were apparently not affected.     

Immunohistochemical localization of 36 and 29 kDa proteins in sparganum.

Antigenic proteins of 36 and 29 kDa were localized in Spirometra mansoni plerocercoid (sparganum) immunohistochemically by avidin biotin complex (ABC) staining. When polyclonal antibodies such as BALB/c mouse serum immunized with crude saline extract of sparganum or confirmed sparganosis sera were reacted as primary antibodies, the positive chromogen (3-amino, 9-ethylcarbazole) reactions were recognized at syncytial tegument, tegumental cells, muscle and parenchymal cells and lining cells of excretory canals. A monoclonal antibody (MAb) which was reacting to 36 and 29 kDa proteins in the extract of the worm was localized at the syncytial tegument and tegumental cells. The present results suggested that the potent antigenic proteins of 36 and 29 kDa in sparganum were produced at the tegumental cells and transported to the syncytial tegument.     

Immunoelectron microscopic localization of partially purified antigens in adult Paragonimus iloktsuenesis.

An immunoelectron microscopy employing immunogold labeling method was performed to detect tissue origin of D1 fraction (D1A) among 5 antigenic protein fractions partially purified by DEAE-anion exchange chromatography from water-soluble crude antigen (PIWA) of adult Paragonimus iloktsuenensis. Immune reactions of adult worm tissues with rabbit serum immunoglobulin immunized with crude antigen (PI-Ig) and D1 antigen (D1-Ig), as well as rat serum immunoglobulin infected with P. iloktsuenensis were observed. D1A showed strong antigenicity in the intestinal epithelium of the worms during the early infection period of 2-4 weeks after infection. The vitellaria also showed stronger antigenicity than the other tissue sites in immune reaction of tissues against all immunoglobulins from 4 to 33 weeks after vitelline development. Therefore, it is suggested that D1A was mainly originated from the intestinal epithelial tissues before the development of vitelline gland of the parasites. Immuno-reactivity of two immunoglobulins (PI-Ig, D1-Ig) was significantly different in intestinal epithelial cytoplasmic protrusions (CP) and intestinal epithelial secretory granules (SG). In the experimental group with D1-Ig, gold particles were labeled significantly in CP than in SG when compared to the PI-Ig group. Thus, the major antigenic materials in D1 antigen having a strong antigenicity in the early infection period was considered to be originated from the intestinal epithelial tissue.     

Fasciola gigantica: Immunolocalization of 28.5 kDa antigen in the tegument of metacercaria and juvenile fluke

Specific monoclonal antibody (MoAb) to 28.5 kDa tegumental antigen (TA) was used to localize this antigen in the tissues of metacercariae, newly excysted juvenile (NEJ), 1, 3, 5, and 7-week-old juveniles of Fasciola gigantica by using indirect immunofluorescence, immunoperoxidase and immunogold techniques. Both indirect immunofluorescence and immunoperoxidase detections showed that this antigen was concentrated in the tegument particularly in its outer rim, tegumental cells and their processes as well as epithelial linings of the oral sucker. Unlike adult F. gigantica, it was not detected in spermatogenic cells in the testes, cells of Mehlis’gland, oocytes within the ovary, and ovum within the egg of parasites. At the ultrastructural level, the immunogold labeling showed deposit of gold particles specifically in G₂ tegumental granules and on the surface membrane. Thus, this 28.5 kDa antigen is expressed in the tegument and associated structures of juvenile parasites, and it could be a major component of the G₂ granules which are shown to fuse with the surface membrane and contribute material to replace the casted-off membrane. This process is the replenishment and turnover of the surface membrane to prevent the attachment of the host immune effector cells.     

Ultrastructural localization of Sm15 and Sm25, two major tegumental adult worm antigens of Schistosoma mansoni.

Sm15 and Sm25 are two of the principal tegumental antigens recognized by antibodies from mice protectively vaccinated with adult worm tegumental membranes and may therefore be potential vaccine candidate antigens. Using antibodies affinity purified from anti-tegumental membrane anti-sera, and antibodies raised against the recombinant antigens, Sm15 and Sm25 were shown to be located specifically in the tegument of adult worms being distributed throughout the syncitium but not associated with the outer membrane.     

Wound healing in the trematode Schistosoma

The ability of adult Schistosoma mansoni to effect wound healing over an exposed surface has been demonstrated. In transected worm segments a new external plasma membrane formed over the exposed tegumental cytoplasm. An elevated leading edge of tegument developed around the margin of the wound; the surface of this region was highly convoluted and there was a proliferation of membranous bodies within its cytoplasm. Inward migration of the leading edge over the exposed internal tissues took place. The resulting new tegument lacked spines and sensory endings. There was no regeneration of basal lamina or tegumentary cytons. In vitro maintenance of worm segments for 3 weeks did not give rise to any major ultrastructural changes in the tissues away from the wound.     

KCl-Extractable surface antigens of Schistosoma mansoni: immunological characterization and applicability in immunodiagnosis

A Schistosoma mansoni antigen preparation was obtained by extraction of adult worms with a 3 M KCl solution. An indirect immunofluorescence reaction on cryostat sections of adult worms showed that the extracted antigens mainly originated from the tegument. The complex antigenic composition of the tegument extract was shown by immunoelectrophoresis against serum from infected mice and immunized rabbits, which gave up to 9 and 17 precipitation lines, respectively. When we compared the use of adult worm antigens and the tegument antigen preparation in the DASS and ELISA tests for immunodiagnosis of human schistosomiasis, the average sensitivity of the tests with the two preparations was about equal, although considerable differences between individual sera occurred. Analysis of tegument antigens, fractionated by gel filtration, showed that the main serological activity of the tegument antigen preparation was due to high molecular weight antigens.     

Ultrastructural study of the host-parasite interface of Moniliformis moniliformis (Archiacanthocephala) in laboratory-infected rats

The fine structure of the host-parasite interface of Moniliformis moniliformis in rats is described, comprising investigations on the ontogenic development of the presomal tegument, on the lesions caused by the worms, and on the host cellular reactions at the points of attachment of the worm. The presomal tegument of the worm contained more fibrous elements than the metasomal tegument. The sclerotization increased with the ages of the worms and toward the tip of the proboscis. The presomal surface coat was more coarsely structured and osmiophilic than that of the metasoma and was neither continuous with the contents of the tegumental crypts nor supported by lipids from necrotic host tissue. The surface coat occasionally detached from the proboscis, probably due to the activity of the surrounding granulocytes of the host. The proboscis hooks lost their tegumental covering during their larval development. Hooks of all worms from rats were invested with a semiliquid lipid coat, apparently derived from tegumental excretions at the base of the hooks. The invaginated area of excretion around the base of the hooks was rich in endoplasmic reticulum. The hooks seemed to renew their lipid coats at certain intervals by dipping into the excreted lipid. In rats all worms, irrespective of their age, attached superficially, penetrating only the intestinal mucosa and the tunica propria. No fibrous connective tissue was found in the lesions caused by the worms, indicating that they frequently changed their site of attachment. At 3-10 days postinfection the host's defense cells observed in the lesions consisted mainly of granulocytes, whereas plasma cells were the predominant leukocytes in lesions of older infections.     

Fasciola hepatica: Glycocalyx replacement in the juvenile as a possible mechanism for protection against host immunity

The response of newly excysted juvenile Fasciola hepatica to immune sheep serum under in vitro conditions was examined using indirect fluorescent antibody labeling and electron microscopy. Flukes acquired a continuous layer of host IgG over the surface during incubation in the presence of antiserum, but when transferred to a medium lacking antiserum they actively sloughed this layer and replaced the former glycocalyx, by a new antigenically similar surface coat. Electron microscope examination of juvenile flukes verified than an immune complex formed at and sloughed from the tegumental surface of those which were incubated in immune serum. T0 secretory bodies produced by the GER/Golgi system of the tegumental cells and stored in the metacercariae were discharged at the apical surface of the tegument, possibly in response to antibody binding. When cycloheximide was included with immune serum in the incubation medium the tegumental cells were unable to synthesize new T0 bodies to replace losses and the number of T0 bodies decreased so that the cytoplasm of the tegumental cells and surface syncytium became virtually devoid of T0 bodies within 48 hr.     

Schistosoma mekongi: the in vitro effect of praziquantel and artesunate on the adult fluke

The efficacy and tolerance of 80 microg/ml praziquantel (PZQ) and 40 microg/ml artesunate (ATS) against adult stage Schistosoma mekongi in vitro were investigated after 3, 6, 12, and 24h incubation by monitoring worm motility and compared tegumental changes using scanning electron microscopy (SEM). Thirty mice were infected with S. mekongi cercaria for 49 days. Adult worms were collected by perfusion method and prepared for in vitro study. Contraction and decreased motor activity were observed after as little as 3h incubation with PZQ and ATS. Some of the worms were immobile 12h after exposure, and died within 24h. The tegument of S. mekongi showed severe swelling, vacuolization and disruption, fusion of the tegumental ridges, collapse and peeling. After 12-24h incubation, PZQ induced similar but they less severe, tegumental changes to those observed after exposure to ATS. The direct observation of the fluke motility and SEM study suggest that ATS is more effective than PZQ in causing tegumental damage in adult S. mekongi, and provides a basis for subsequent clinical trials.     

Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm

Hockley D. J. and McLaren D. J. 1973. Schistosoma mansoni: changes in the outer membrane of the tegument during development from cercaria to adult worm. International Journal for Parasitology3: 13–25. The tegumental outer membrane of the cercaria is trilaminate: the adult worm, however, has a seven-layered membrane. Formation of the heptalaminate membrane commences immediately after the cercaria has penetrated the vertebrate host: multilaminate membrane-bounded vacuoles are passed from subtegumental cells into the tegument where they enlarge, join to the outer membrane and open to the exterior. The heptalaminate limiting membrane of the vacuole thus becomes the outer membrane of the tegument. At the same time the original trilaminate tegumental membrane is formed into microvilli which are cast off and thus the cercarial outer membrane is lost. Schistosomula usually have a heptalaminate outer membrane within three hours of penetration. After this time the large vacuoles are replaced by smaller membraneous bodies which presumably contribute to the outer membrane during growth of the schistosomulum. The membraneous bodies are also present in the tegument of the adult worms and there is some evidence that the outer membrane is continually renewed.     

Effects of praziquantel and artesunate on the tegument of adult Schistosoma mekongi harboured in mice

The effects of praziquantel and artesunate on the tegument of adult Schistosoma mekongi harboured in mice were compared using scanning electron microscopy (SEM). Forty-two mice infected with S. mekongi for 49 days were treated intragastrically with either 300 mg/kg praziquantel or 300 mg/kg artesunate. Mice were sacrificed 1 or 3 days post-treatment. Worms were collected by perfusion and examined by SEM. One to 3 days after administration of artesunate, the tegument of S. mekongi showed severe swelling, vacuolization, fusion of the tegumental ridges and loss or shortening of the spines on the trabeculae, collapse and peeling. Praziquantel induced similar tegumental alterations as those observed after administration of artesunate, but they were less severe. Three days post-treatment, there was evidence of recovery only in the case of praziquantel. The results of our study suggest that artesunate is more effective than praziquantel in causing tegumental damage in adult S. mekongi, and provides a basis for subsequent clinical trials.     

Characterization of proteins and immunogens released by adult Schistosoma mansoni

The proteins released in vitro by metabolically radiolabeled adult Schistosoma mansoni were identified by 2-dimensional gel electrophoresis. To determine the origin of these proteins, adult worms were fractionated into surface membrane, tegument, and remaining body components, and the electrophoretic patterns of the proteins in the 3 fractions were compared to those of the released proteins. The immunogens present in these fractions then were identified by immunoprecipitation with sera from humans infected with S. mansoni. This analysis indicated that essentially all of the proteins released from the worm were immunogenic, whereas most of the major membrane and tegumental proteins were not reactive with the immune sera. Thus, it appears that the adult worm is defended against immune attack by detection of the host's antibody response against released proteins rather than against proteins-exposed on the worm's surface.     

Fasciola hepatica: IgG and IgA levels in the serum and bile of infected cattle

Bile and serum samples were collected from calves with an implanted cannula throughout a 20-week period of infection with Fasciola hepatica. Using indirect fluorescent antibody labelling and plastic-embedded sections of juvenile and adult flukes as antigens, estimates were made of the relative concentrations of IgG and IgA specific for fluke tegumental and gut antigens in the samples of serum and bile. In serum, antibodies against juvenile (t1) tegument and gut antigens reached peak concentrations 4–6 weeks postinfection and declined slowly thereafter as flukes became established in the bile ducts. IgG against adult tegument (t2) antigens appeared in the serum 6 weeks after infection, but no IgA against t2 was detected. In the bile, both IgG and IgA titres against t1 and gut antigens rose to peak values at 4–6 weeks after infection, but there was no activity against t2 antigen. The Ig levels in bile were considerably lower than in serum. Much more IgA relative to IgG occurred in bile as compared to serum (IgG/IgA ratio in serum was 16–32, in bile 1–2) suggesting a role for IgA in defence at mucosal surfaces. Comparison of the antibody profiles in bile and serum suggested that IgG in the bile was derived from circulating IgG whereas IgA may have been preferentially concentrated in the bile.     

Detection of antibodies to Angiostrongylus cantonensis in serum and cerebrospinal fluid of patients with eosinophilic meningitis

Adult and young adult antigens of Angiostrongylus cantonensis were purified by immuno-affinity chromatography and used to detect antibody in serum and cerebrospinal fluid (CSF), by enzyme-linked immunosorbent assay (ELISA), in cases of human eosinophilic meningitis or meningoencephalitis. The levels of IgG, IgA, IgM and IgE antibodies to A. cantonensis in these patients were higher than levels in control subjects. Antibodies in patients detected against adult and young adult worm antigens of A. cantonensis did not differ significantly. Significantly higher IgM and IgE antibody levels were observed in serum compared with CSF from infected patients (Student's t-test, P less than 0.05). Both adult and young adult A. cantonensis antigens proved to be highly sensitive in ELISA for serum antibodies; however, the sensitivity was significantly lower in tests on CSF.